Alginate and other hydrogels are attractive materials for a variety of biomedical applications, including cell transplantation, and drug delivery. In many of these applications one desires to either seed cells into the material, or allow for cellular invasion following implantation into the body. However, alginate is typically used in the physical form of a hydrogel, with small pores (nm size scale) that do not allow for cell movement in or out of the material. This invention is directed to a new approach to form porous hydrogel materials by first creating gas pockets in the gel and then removing this gas. The removal of the gas creates a porous material, and the initial incorporation of sufficient gas allows one to create a material with an open, interconnected pore structure. Advantageous features of the resulting materials, in addition to their interconnected pore structure, may include that the pore structure is maintained over extended time periods and that the gels maintain a high mechanical integrity that allows seeding with cells and implantation without destruction or compression of the material.
The invention is in contrast to other processing approaches typically used to achieve a porous structure with these types of materials (e.g., lyophilization) in which the porous nature is lost as the material rehydrates and/or the material is significantly weakened by the process.
An approach to form and subsequently remove gas bubbles from alginate gels has been previously described (Gotoh et al., Cytotechnology 11, 35 (1993)). However, the methods described in this article did not lead to the formation of structures with a sufficient degree of porosity or a sufficiently open interconnected pore structure.
The method described herein is a considerable modification of the Gotoh et al. method and is conducted under conditions outside of the ranges described therein. An object of the invention is to provide biocompatible hydrogel materials, for example alginate materials, which have a significantly macroporous and open pore structure, e.g., such that the pores are sufficiently open and sized to allow cellular transport therein. This facilitates vascularization and structural integration with the surrounding tissue when used in tissue engineering applications. Thus, the macroporous hydrogel will preferably have pores of at least 1 xcexcm, particularly from 10 to 1000 xcexcm. While not limited thereto, the overall porosity is preferably from 30 to 90%, more preferably 35 to 75%. The total surface accessible interconnected porosity is preferably from 30-80%, more preferably 35-70%.
Upon further study of the specification and appended claims, further objects and advantages of this invention will become apparent to those skilled in the art.
Objects according to the invention can be achieved by a method for preparing a hydrogel material having macroporous open pore porosity, which comprises:
a) providing a solution of a hydrogel-forming material, a surfactant and, optionally, a gas-generating component which solution is capable of being mixed in the presence of a gas (either added or generated by the gas-generating component) to incorporate the gas in the solution and form a stable foam;
b) forming a stable foam by mixing the solution in the presence of a gas and/or, if the gas-generating component is present, by subjecting the solution to conditions or agents which result in generation of gas from the gas-generating component;
c) exposing the stable foam to conditions and/or agents which result in gelling of the hydrogel-forming material to form a hydrogel containing gas bubbles therein;
d) releasing the gas bubbles from the hydrogel, for example by subjecting it to a vacuum, to form a hydrogel material having macroporous open pore porosity.
Steps b) and c) may be performed simultaneously or in series.
Any hydrogel-forming material which can provide the desired effect of resulting in a foam which allows preparation of the open pore material can be used in the invention. Examples of materials which can form hydrogels include polylactic acid, polyglycolic acid, PLGA polymers, alginates and alginate derivatives, gelatin, collagen, agarose, natural and synthetic polysaccharides, polyamino acids such as polypeptides particularly poly(lysine), polyesters such as polyhydroxybutyrate and poly-xcex5-caprolactone, polyanhydrides; polyphosphazines, poly(vinyl alcohols), poly(alkylene oxides) particularly poly(ethylene oxides), poly(allylamines)(PAM), poly(acrylates), modified styrene polymers such as poly(4-aminomethylstyrene), pluronic polyols, polyoxamers, poly(uronic acids), poly(vinylpyrrolidone) and copolymers of the above, including graft copolymers.
A preferred material for the hydrogel is alginate or modified alginate material. Alginate molecules are comprised of (1-4)-linked xcex2-D-mannuronic acid (M units) and (xcex1-L-guluronic acid (G units) monomers which vary in proportion and sequential distribution along the polymer chain. Alginate polysaccharides are polyelectrolyte systems which have a strong affinity for divalent cations (e.g. Ca+2, Mg+2, Ba+2) and form stable hydrogels when exposed to these molecules. See Martinsen A., et al., Biotech. and Bioeng., 33 (1989) 79-89. Calcium cross-linked alginate hydrogels have been used in many biomedical applications, including materials for dental impressions (Hanks C. T.,et al., Restorative Dental Materials; Craig, R. G., ed., Ninth Edition, Mosby (1993)), wound dressings (Matthew I. R. et al., Biomaterials, 16 (1995) 265-274), an injectable delivery medium for chondrocyte transplantation (Atala A., et al., J Urology, 152 (1994) 641-643), and an immobilization matrix for living cells (Smidsrod O., et al, TIBTECH 8 (1990) 71-78).
An alternative embodiment utilizes an alginate or other polysaccharide of a lower molecular weight, preferably of size which, after dissolution, is at the renal threshold for clearance by humans. Preferably, the alginate or polysaccharide is reduced to a molecular weight of 1000 to 80,000 daltons, more preferably 1000 to 60,000 daltons, particularly preferably 1000 to 50,000 daltons. It is also useful to use an alginate material of high guluronate content since the guluronate units, as opposed to the mannuronate units, provide sites for ionic crosslinking through divalent cations to gel the polymer.
Alginate can be xcex3-irradiated in a controlled fashion to cause a random fission of the polymer chains and generation of appropriate low molecular weight alginate fragments [Hartman et al., Viscosities of cacia and sodium alginate after sterization by cobald-60. J. Pharm. Sci.; 1975, 64(5): 802-805; King K., Changes in the functional properties and molecular weight of sodium alginate following xcex3-irradiation. Food Hydrocoll. 1994; 8(2): 83-96; Delincxc3xa9e H., Radiolytic effects in food. In: Proceedings of the international workshop on food irradiation. 1989, p 160-179]. In these earlier descriptions of the degradation of alginate utilizing xcex3-irradiation, the conditions used were outside the range required to generate materials with molecular weights lower than 200 kD, or they were used on alginate solutions rather than the bulk material. Other methods for the controlled degradation of alginate are also available [Kimura et al., Effects of soluble alginate on cholesterol excretion and glucose tolerance in rats. J. Ethnopharn.; 1996, 54: 47-54; Purwanto et al., Degradation of low molecular weight fragments of pectin and alginates by gamma-irradiation. Acta Alimentaria; 1998, 27(1): 29-42], but xcex3-irradiation is a reliable and simple technique for generating low molecular alginates. The reduction in molecular weight can also be effected by hydrolysis under acidic conditions or by oxidation, to provide the desired molecular weight. The hydrolysis may be conducted in accordance with a modified procedure of Haug et al. (Acta. Chem. Scand., 20, p. 183-190 (1966), and Acta. Chem. Scand., 21, p. 691-704 (1967)), which results in a sodium poly(guluronate) of lower molecular weight which is essentially absent of mannuronic acid units. The oxidation to lower molecular weight is preferably conducted with a periodate oxidation agent, particularly sodium periodate; see PCT/US97/16890.
As expected, alginate solutions prepared from lower molecular weight alginates possessed decreased viscosities, and more concentrated solutions were required to form stable foams. The difference in the total void volume of beads formed from low and high molecular weight alginate is likely caused by the increased alginate concentration required to form stable foams in the low molecular weight alginates.
For alginate materials, it is preferred to use starting solutions of alginate salt in an amount, for example, of 3 to 10% w:w (weight based on weight of water), more preferably 3 to 5% w:w. For other materials,. the amount used in the starting solution will depend upon the material used, however, it is preferred to use at least 3% w:w in the starting solution. This will preferably result in concentrations of the alginate or other hydrogel-forming material in the solution to be foamed of more than 3% weight, particularly 3-10%, more particularly, 3-5%.
The hydrogel-forming material is the precursor, ungelled form of the hydrogel. It will be a soluble form of the hydrogel which is capable of being gelled by application of some condition or agent. For example, alginate salts, such as sodium alginate, are gelled in the presence of divalent cations, such as calcium present in calcium chloride. Other materials may be gellable by a change in pH or temperature, for example.
As the surfactant, any surfactant which will facilitate formation and stabilization of gas bubbles in the solution,without preventing the other steps of the method, can be used. Useful examples thereof include bovine serum albumin (BSA), the pluronic class of surfactants (e.g., F108 and F68), polyethylene glycol and propylene glycol alginate surfactants. The amount of surfactant used will depend on the amount and type of the hydrogel being formed and an amount which facilitates formation and stabilization of gas bubbles in the solution, without preventing the other steps of the method, can be used.
Particularly preferred are the pluronics surfactants, as the bovine albumin originally used may elicit a foreign body response in vivo. Pluronics are non-ionic surfactants and their foaming properties increase with increasing ethylene oxide content. A similar trend is observed as the molecular weight of the hydrophobic portion increases at a fixed ethylene oxide content (Alexandridis et al., Micellization of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymers in aqueous solutions: thermodynamics of copolymer association. Macromolecules; 1994, 27: 2414-2425.) The particular pluronics, F108 and F68, possess the same ethylene oxide content (80% (w/w)) but differ in average molecular weight (14,600 and 8,400 Daltons, respectively). The increased porosity of beads formed with F108 is consistent with its foaming ability as compared to beads formed with F68. It is also known that pluronics surfactants do not micellize at a critical micelle concentration (CMC) but instead aggregate over a wide range of concentrations (ACR). The limiting aggregation concentration (LAC) is the point at which the surfactant reaches saturation, which corresponds to the more conventional CMC. The ACR for F108 ranges from 400-50,000 ppm (see Alexandridis cited above). The LAC or CMC for this surfactant has been reported to be  greater than 50,000 ppm or 4.5% w/v, respectively (see Aexandridis cited above). Thus, it seems plausible that a high surfactant concentration can lead to premicellar aggregates, which directly effect the porosity determined in the beads.
In a particularly preferred embodiment, the hydrogel-forming material is a low molecular weight alginate and the surfactant is a pluronics surfactant.
For use of BSA as the surfactant, the method may be conducted using a BSA concentration in the final solution to be foamed of 0.05-1% weight. The amount used will depend on the amount of the algifiate or other hydrogel material used. A BSA stock solution of 1 to 10% weight BSA in water can be used for this purpose. For example, using 2 grams of a 3 to 5% w:w of alginate, 240 to 400 mg of a 15% solution of BSA has been found useful. The weight ratio of BSA to alginate may be from 1:10 to 1:60, preferably 1:10 to 1:20, for some applications, but it is not limited thereto.
If a gas-generating component is provided, it is preferred to use sodium bicarbonate, which will release carbon dioxide gas when exposed to a mild acid, for example, acetic acid. For this purpose, a 10% volume acetic acid solution may be used which provides at least an amount of acetic acid equimolar to the amount of carbon dioxide to be released from the sodium bicarbonate. For example, the bicarbonate can be added in powder form or as a bicarbonate solution of 1.0M to 2.0M to provide a concentration of 0.5 to 5.0% weight in the solution to be foamed. When using an alginate hydrogel and a BSA surfactant, it has further been found that the ratio of BSA to bicarbonate has an effect on the product. It is preferred in this case that the weight ratio of 15% weight BSA solution to the 1.0 to 2.0M bicarbonate solution is 2:1 to 1:1. Other materials which release gases upon application of some condition or agent may be used provided they will result in formation of gas bubbles in the hydrogel which are releasable upon application of a vacuum and do not otherwise interfere in the preparation.
A gas-generating component may not be necessary if the solution of hydrogel-forming material and surfactant can be mixed in the presence of gas to provide suitable gas bubbles in the resulting hydrogel. Preferably the solution is mixed in the presence of air to result in the foaming and subsequent formation of air bubbles when the hydrogel is gelled. When a gas-generating component is used, the hydrogel may have gas bubbles of air provided by the mixing as well as gas bubbles generated by the gas-generating component. Any mixing means which results in adequate foaming can be used.
The stable foam resulting from mixing of the above-described solution is gelled in a manner dependent upon the hydrogel-forming material, e.g., by contact with a gelling agent or a change in pH or temperature. For alginate hydrogels, the gelling is effected by contact with divalent cations in solution, e.g., a calcium chloride solution of from 0.1 to 1.0M, preferably about 0.5M. The divalent cations serve to jonically crosslink the alginate. The manner of exposing the solution to the gelling agent or condition will depend on the desired shape of resulting porous hydrogel material. For example, hydrogel beads can be provided by adding the stable foam dropwise to a solution of the gelling agent, such as through a syringe or a syringe pump for scaled up applications. In a similar manner, the stable foam may be provided continuously through a syringe device to provide the porous hydrogel in a fibrous form. The stable foam may also be cast in a desired shape and subject to the gelling agent or gelling condition to provide a shaped article, which may be particularly useful for tissue regeneration applications. Other forms of the material may be prepared using means available in the art.
The resulting hydrogel will contain gas bubbles and exposure thereof to a vacuum will draw out the entrapped gas bubbles to create an open pore macroporous hydrogel.
In one preferred embodiment, the process involves first providing a solution of sodium alginate in water. Sodium bicarbonate and bovine serum albumin (BSA) are then added to this solution and mixed to allow for incorporation of air bubbles in the resulting solution to create a stable foam. This solution is then placed in a syringe and extruded dropwise into a stirred solution of calcium chloride and acetic acid in water (gelling solution). The calcium ions serve to gel the alginate, while the acetic acid reacts with the bicarbonate to generate carbon dioxide gas in the hydrogel. The gelled alginate in the form of microbeads is collected separately from the solution. The alginate is subsequently exposed to a vacuum to draw out the entrapped gas bubbles (both of air and carbon dioxide) and create the open pore structure.
It is important to note that as the hydrogel is varied the specific, optimal conditions may vary as well. For example, utilization of a lower molecular weight alginate will decrease the solution viscosity, necessitating a higher alginate concentration and/or higher surfactant concentration, and vice versa. Varying the guluronic acid content of the alginate will alter the strength of the hydrogel and require either an increased vacuum to remove gas bubbles or a decreased starting. alginaie concentration.
The materials prepared by the process of the invention exhibit a wide range of utilities. They may be applied to any use which requires a porous hydrogel material, particularly with an open pore structure.: For instance, the materials are useful as matrices or scaffolds into which cells can migrate, the cells being compatible therein and growing to achieve their intended function, such as in tissue replacement, eventually replacing the matrix depending on its biodegradability. Furthermore, the materials can be used to provide matrices already bound to cells, which may then be surgically implanted into a body. Further, the materials can be used as a sustained release drug delivery system, as wound healing matrix materials, as matrices for in vitro cell culture studies or uses similar thereto. The stable structure of the materials of the invention provides ideal cell culture conditions.
The materials of the invention may also have application in cell transplantation, including for hepatocytes (see, D. J. Mooney, P. M. Kaufmann, K. Sano, K. M. McNamara, J. P. Vacanti, and R. Langer, xe2x80x9cTransplantation of hepatocytes using porous biodegradable sponges,xe2x80x9d Transplantation Proceedings, 26, 3425-3426 (1994); D. J. Mooney, S. Park, P. M. Kaufrnann, K. Sano, K. McNamara, J. P. Vacanti, and R. Langer, xe2x80x9cBiodegradable sponges for hepatocyte transplantation,xe2x80x9d Journal of Biomedical Materials Research, 29, 959-965 (1995)), chondrocytes and osteoblasts (see, S. L. Ishaug, M. J. Yaszemski, R. Biciog, A. G. Mikos; xe2x80x9cOsteoblast Function on Synthetic Biodegradable Polymersxe2x80x9d, J. of Biomed. Mat. Res., 28, p. 1445-1453 (1994)).
Smooth muscle cells may readily adhere to the material prepared according to the invention and create three-dimensional tissues especially if appropriate cell adhesion ligand are coupled to the hydrogel structure within these porous structures; thus, they provide a suitable environment for cell proliferation. In addition, these materials have potential to incorporate growth factors.
Another useful application for the polymer matrices of the invention is for guided tissue regeneration (GTR). This application is based on the premise that progenitor cells responsible for tissue regeneration reside in the underlying healthy tissue and can be induced to migrate into a defect and regenerate the lost tissue. A critical feature of materials for GTR is the transport of cells into the material, a property which is dictated by the pore size distribution and pore continuity, i.e., interconnectivity. The material must allow the desired cells to invade the material while preventing access to other cell types.
The entire disclosure of all applications, patents and publications, cited above and below, is hereby incorporated by reference.
In the foregoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius; and, unless otherwise indicated, all parts and percentages are by weight.